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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
3' end has lots of gt repeats - had to make large products and mut probe is across a string of 5g's which im not thrilled about.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Mutant= 96 bp
Wild Type =138 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 31973 | CCA CAT CCT ACC CTT TCC AC | Common | A | |||
| 31975 | Fluorophore-1 | CCC TGA GGG GGT GAA TAA AT | Quencher-1 | WT Probe | ||
| 31980 | CTC CCC ACT GAA CAG ACA CA | Wild type Reverse | A | |||
| 33697 | Fluorophore-2 | ACT GTC AGA GAC GGG GGT G | Quencher-2 | MUT Probe | ||
| 33698 | CCA AGC AGT TAG CAC CAC AC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 31973 | 0.40 uM |
| 31980 | 0.40 uM |
| 33698 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.